SYSTEMATIC REVIEW published: 15 April 2021 doi: 10.3389/fbioe.2021.622524

Foreign Body Reaction to Implanted Biomaterials and Its Impact in Nerve Neuroprosthetics

Alejandro Carnicer-Lombarte 1, Shao-Tuan Chen 1, George G. Malliaras 1* and Damiano G. Barone 1,2*

1 Electrical Engineering Division, Department of Engineering, University of Cambridge, Cambridge, United Kingdom, 2 Division of Neurosurgery, Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom

The implantation of any foreign material into the body leads to the development of an inflammatory and fibrotic process—the foreign body reaction (FBR). Upon implantation into a tissue, cells of the immune system become attracted to the foreign material and attempt to degrade it. If this degradation fails, fibroblasts envelop the material and form a physical barrier to isolate it from the rest of the body. Long-term implantation of medical devices faces a great challenge presented by FBR, as the cellular response disrupts the interface between implant and its target tissue. This is particularly true for nerve

Edited by: neuroprosthetic implants—devices implanted into nerves to address conditions such as Elisa Castagnola, sensory loss, muscle paralysis, chronic pain, and epilepsy. Nerve neuroprosthetics rely on University of Pittsburgh, United States tight interfacing between nerve tissue and electrodes to detect the tiny electrical signals Reviewed by: carried by axons, and/or electrically stimulate small subsets of axons within a nerve. Jennifer Patterson, Instituto IMDEA Materiales, Spain Moreover, as advances in microfabrication drive the field to increasingly miniaturized PaYaM ZarrinTaj, nerve implants, the need for a stable, intimate implant-tissue interface is likely to Oklahoma State University, United States quickly become a limiting factor for the development of new neuroprosthetic implant *Correspondence: technologies. Here, we provide an overview of the material-cell interactions leading George G. Malliaras to the development of FBR. We review current nerve neuroprosthetic technologies [email protected] (cuff, penetrating, and regenerative interfaces) and how long-term function of these Damiano G. Barone [email protected] is limited by FBR. Finally, we discuss how material properties (such as stiffness and size), pharmacological therapies, or use of biodegradable materials may be exploited Specialty section: to minimize FBR to nerve neuroprosthetic implants and improve their long-term stability. This article was submitted to Biomaterials, Keywords: foreign body reaction, nerve neuroprosthetics, neural implants, neural interface, peripheral nerve a section of the journal stimulation, biocompatibility Frontiers in Bioengineering and Biotechnology Received: 28 October 2020 INTRODUCTION Accepted: 19 March 2021 Published: 15 April 2021 Implantable devices constitute a class of treatments for disease or dysfunction with unique Citation: therapeutic potential. By remaining implanted for long periods of time while delivering a therapy Carnicer-Lombarte A, Chen S-T, directly into the affected tissue, implants can target the source of a dysfunction with great accuracy Malliaras GG and Barone DG (2021) and for as long as required. One of the most versatile class of implants are nerve neuroprosthetics: Foreign Body Reaction to Implanted implantable devices which interface with the peripheral nerves of the body. Nerves serve as Biomaterials and Its Impact in Nerve communication bridges between the brain and all peripheral structures, transmitting information Neuroprosthetics. Front. Bioeng. Biotechnol. 9:622524. between the two. Whether by activating nerves through electrical stimulation or by reading doi: 10.3389/fbioe.2021.622524 their electrical signals, nerve neuroprosthetics offer a myriad of applications such as the control

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 April 2021 | Volume 9 | Article 622524 Carnicer-Lombarte et al. FBR in Nerve Neuroprosthetics of bladder function (Brindley et al., 1982), management of isolates the implanted material. When the foreign material is depression (Nemeroff et al., 2006), and restoration of sensory implanted with the aim of delivering a therapy, both the acute perception in amputees (D’Anna et al., 2019). While implantable (predominantly inflammatory) and chronic (fibrotic) stages of technologies—and in particular nerve neuroprosthetics—have FBR pose significant challenges to its integrity and therapeutic extensively evolved over the last few decades by drawing on function. The complex timeline of FBR is summarized in advancements in biomaterials and manufacturing techniques, the Figure 1, and exemplified in Figure 2. clinical use of implants continues to be severely hampered by the challenges arising from the hostile environment of the body. Acute FBR The aim of this review is to highlight one of the major The acute phase of FBR begins immediately after implantation. challenges to the clinical translation of implantable materials and The tissue damage and extravasation of blood which inevitably devices: the foreign body reaction (FBR). The slow onset of this occurs during the implantation process triggers an immediate reaction triggered by the body and its dynamic profile (beginning rush of inflammatory-mediating cells to the area. Within with an acute inflammatory attack and transitioning to a long- seconds of implantation, proteins—many of which are derived term fibrotic response) make it difficult to predict and test from extravasated blood, such as albumin and fibrinogen— for during the design and development of implantable devices. become non-specifically adsorbed to the surface of the implant Research often focuses on the optimisation of implantable (Figure 1—protein binding). This layer of proteins becomes a technologies for good function over a period of days to provisional matrix, through which cells gathering in the area can weeks after implantation. While it is impractical to test new identify and interact with the foreign body. As time progresses, technologies for longer periods of time at every step, clinical proteins of this provisional matrix undergo a dynamic process devices have to remain implanted in human patients for years of adsorption-desorption, through which the smaller proteins or decades. In order to facilitate bench-to-bedside translation which are initially found around the implant surface (such of new technologies, it is therefore crucial to consider the as albumin) are progressively replaced by larger ones. This effects that FBR will have on both the implanted device and the process—known as the Vroman effect—can vary in protein surrounding tissue. composition and time course for different implanted materials, In this review we present an overview of the cellular leading to differences in FBR across different materials even at events leading to and maintaining FBR in order to provide this early stage (Wilson et al., 2005; Xu and Siedlecki, 2007). an understanding of the processes involved in this reaction The implantation process marks the beginning of a cascade and the challenges presented to implanted devices, and discuss of cellular events that make up FBR. Within minutes of some of the general design principles that can be followed implantation, neutrophils—the early responders in any type of to minimize the impact of FBR. We discuss the impact of tissue injury—migrate into the area. Neutrophils adhere to the FBR in the emerging field of nerve neuroprosthetics—how protein layer surrounding the implant and begin to release nerve neuroprosthetic designs have evolved over the years and factors which promote the progression of the inflammatory addressed the challenges posed by FBR. Finally, we look at the process (such as reactive oxygen species and proteolytic enzymes, future of the field by discussing novel strategies being developed Figure 1—neutrophil recruitment, Figure 2). Together with to combat FBR to implants, and how these may be implemented similar chemical signals resulting from blood clotting and in nerve neuroprosthetic devices. mast cell activation, these factors increase vascular permeability and attract monocytes into the site of implantation. Once arrived, monocytes begin to differentiate into macrophages, IMPLANTATION OF BIOMATERIALS AND which in turn proliferate and populate the lesion (Figure 1– THE FOREIGN BODY REACTION monocyte recruitment and differentiation to macrophages, Figure 2). Within two days of implantation the initial wave Foreign body reaction (FBR) is an unavoidable process which of neutrophils has completely disappeared to give way to a takes place whenever any material becomes implanted into population of macrophages (Anderson et al., 2008; Franz et al., the body. The process of implantation injures the tissue 2011), which self-sustains itself by continuously proliferating around the foreign object, which triggers an inflammatory and releasing chemoattractants that recruit further macrophages. process. Over a period of weeks to months this inflammatory These macrophages also mediate the core of the inflammatory process develops into a fibrotic response, which envelops and response, releasing pro-inflammatory actors such as TNFα (tumor necrosis factor α), and interleukins IL-1b, IL-6, and IL- ′ Abbreviations: CNS, Central nervous system; DAPI, 4 ,6-diamidino-2- 8 (Jones et al., 2007; Mesure et al., 2010). Up to this stage the phenylindole; ECM, Extracellular matrix; FBGC, Foreign body giant cell; FBR, inflammatory response to device implantation is very similar to Foreign body reaction; FINE, Flat interface nerve electrode; IL-1β, Interleukin 1 beta; LIFE, Longitudinally implanted intrafascicular electrode; NF, Neurofilament; that occurring in any injury—neutrophils rush to the area and PDGF, Platelet-derived growth factor; PDMS – Polydimethylsiloxane; PET, recruit macrophages, which first eliminate invading threats and Polyethylene terephthalate; PLA, Poly(lactic acid); PLGA, Poly(lactic-co-glycolic then mediate tissue repair. However, as macrophages populate acid); PMMA, Poly(methyl methacrylate); PNS, Peripheral nervous system; ROS, the site of implantation, this initial acute inflammatory response Reactive oxygen species; SEM, Scanning electron microscope; SPINE, Slowly penetrating interfascicular nerve electrode; TGF-β, Transforming growth factor develops into FBR. beta; TIME, Transverse intrafascicular multichannel electrode; TNFα, Tumor As macrophages populate the lesion site they begin to necrosis factor alpha; VEGF, Vascular endothelial growth factor. adhere to and cover the exposed surface of the implant. This

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 2 April 2021 | Volume 9 | Article 622524 Carnicer-Lombarte et al. FBR in Nerve Neuroprosthetics

FIGURE 1 | Timeline of the events leading to the development of the foreign body reaction to a material following its implantation into the body. The composition of the cell population adhered to the surface of the implant evolves over time following the initial implantation. Factors released by cells (indicated by blue text) contribute to the recruitment of further cells and progression of FBR. ROS, reactive oxygen species. adhesion process is thought to be a critical component of fragments. The factors released by macrophages as a result FBR initiation, and occurs through integrins—a broad class of this frustrated phagocytosis include degrading enzymes and of transmembrane proteins which bind to other proteins of reactive oxygen species (Hansen and Mossman, 1987; Anderson, the tissue environment—that specifically bind to the proteins 2001). This macrophage attack poses a significant challenge adsorbed to the implant’s surface. In particular, αMβ2 integrin to implant stability. Otherwise stable biomaterials can exhibit is considered crucial for this initial adhesion stage as it surface degradation and cracking when exposed to the hostile specifically binds to serum proteins of the implant surface inflammatory environment created by macrophages (Labow such as fibronectin and fibrinogen (Anderson et al., 2008). et al., 2001; Wiggins et al., 2001), which may cause a breakdown Following this initial adhesion stage macrophages undergo of the implant or the leaching of toxic species into the tissue from cytoskeletal remodeling. Bound macrophages flatten over the the underlying layers. surface of the implant in an attempt to engulf and phagocytose If macrophages successfully degrade and phagocytose the it, and extend podosomes—structures specialized in proteolysis implant during this acute phase of FBR, the reaction ends, and extracellular matrix remodeling. Bound and activated and the tissue slowly returns to normal. Some implantable macrophages also secrete chemoattractive factors which continue devices are designed to exploit this inflammatory process to recruit macrophages even after the initial implantation injury to degrade after they have carried out their therapeutic has resolved (Crowe et al., 2000). role. A common example of this are regeneration conduits: The macrophage layer forming around the implant creates implants inserted into lesioned tissue designed to guide tissue a defined and isolated space. Being unable to phagocytose regrowth and be degraded as the tissue heals (Arslantunali the entire implant due to its large size, macrophages instead et al., 2014). In many cases, however, implants are required secrete factors into this space in an attempt to break to remain implanted indefinitely to continue carrying out down the foreign body and instead phagocytose the resulting their therapeutic role. When in these cases macrophages

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FIGURE 2 | Foreign body reaction against intraperitoneally-implanted PMMA beads (125–180 µm). Reproduced under the terms of the Creative Commons Attribution License (Christo et al., 2016). Progression from acute to chronic FBR stages can be observed in counts of cells surrounding implants (A-C) and in the thickening of tissue capsule around them (H). (A–C) Quantification of cell numbers in the peritoneum following bead implantation, of all cells (A), neutrophils (B,C) (i), macrophages (B,C) (ii). (D) Explanted PMMA beads. Fibrotic encapsulation of beads visible at longer time points (white arrows). (E–G) Individual explanted beads stained for adhered leukocytes using Diff-Quik staining. (H) Bead aggregates sectioned and stained via immunohistochemistry for albumin and fibrinogen (brown staining). Scale bar: 250 µm.

fail to degrade the implant, FBR transitions into its barrier between the implant and the host tissue into which it was chronic stage. implanted, and can greatly difficult the delivery of any therapy into the surrounding tissue. FBR progressively transitions into its Chronic FBR chronic fibrotic stage over the course of weeks after implantation The chronic phase of FBR is characterized by a transition from and, unless the implant is destroyed or removed, it remains solely inflammatory to a fibrotic process. In contrast to the active indefinitely. direct challenges to an implant’s survivability posed by acute During the transition to the chronic stage of FBR macrophages FBR, the chronic stage of FBR involves the encapsulation of the switch from a pro-inflammatory activation phenotype (M1 implant in a layer of fibrous tissue. This fibrotic layer acts as a macrophages) to an anti-inflammatory and tissue generation

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 4 April 2021 | Volume 9 | Article 622524 Carnicer-Lombarte et al. FBR in Nerve Neuroprosthetics phenotype (M2 macrophages) (Mantovani et al., 2004; Lawrence capable of regrowing if damaged or removed, and can adapt if and Natoli, 2011; Sridharan et al., 2015). This phenotypic the properties of the enclosed implant change. transition is often observed as part of the normal wound healing It is worth noting that the described process of FBR can process, marking the switch from the elimination of foreign differ in certain tissues. The most notable example is the central threats in an injury to the healing of the tissue (Brown et al., 2012; nervous system. Due to the tight control the body exerts Hesketh et al., 2017). During FBR, however, M2 macrophages over cell and molecule crossing from the bloodstream into instead play a role in the formation of a fibroblast and ECM-rich the central nervous system, FBR here is driven by different capsule that covers and isolates the implant. cell types. Namely, inflammation is mediated by CNS-resident M2 macrophages become central orchestrators of fibrosis microglia (rather than macrophages), while fibrosis is driven by during FBR, attracting and organizing fibroblasts to the implant’s astrocytes instead of fibroblasts (Polikov et al., 2005; Salatino surface. M2 macrophages decrease inflammatory activity by et al., 2017). Despite these differences, the general principles releasing anti-inflammatory cytokines such as IL-10 (Klopfleisch, underlying FBR and the consequences (formation of a fibrotic 2016). TGF-β released by these macrophages attracts local capsule, inflammatory damage to the surrounding tissue) still fibroblast populations, and induces their activation on arrival apply (Polikov et al., 2005; Salatino et al., 2017). Despite the (Ignotz and Massagué, 1986). These activated fibroblasts similarities in tissue composition and function that the central adhere to the implant’s surface and begin depositing layers of and peripheral nervous systems share, FBR in peripheral nerves extracellular matrix proteins (Figure 1—fibrotic encapsulation). is not driven by microglia and astrocytes, but by macrophages The activation of fibroblasts involves their transdifferentiation and fibroblasts like in most other tissues. into myofibroblasts—a cell type also responsible for the The FBR capsule poses a significant challenge to the formation of scar tissue and commonly found in wound healing therapeutic function of implantable devices. While the chronic (Hinz et al., 2007). PDGF—also released by macrophages— stage of FBR involves a less aggressive immune attack to induces the proliferation of myofibroblasts, which over time the implanted materials, the growing fibrotic capsule forms cover the entire surface of the implant (Bonner, 2004). a physical barrier that interferes with the delivery of agents A hallmark of FBR is also the fusion of macrophages into or sensing of signals in the surrounding tissue. While certain polynucleate foreign body giant cells (FBGCs) on the surface of classes of implants—such as those carrying out a structural the implant. Within days of implantation macrophages adhered role, or delivering large amounts of therapeutic agents to large to the implant begin to fuse with each other into much larger volumes of tissue—are less affected by the FBR capsule, its cells (Figure 1—foreign body giant cell formation), capable of presence is an increasingly relevant problem. As implantable phagocytosing larger particles (> 10 µm, in contrast to the technologies continue to progress toward more refined devices < 5 µm that macrophages can phagocytose) (Anderson et al., capable of interacting with ever smaller subsets of tissue, the 2008). The dynamics of the fusion process of macrophages into disruption of the tissue-implant interface by FBR becomes a FBGCs appears to be tightly tied to the composition of the layer leading cause of failure (Salatino et al., 2017; Spearman et al., of proteins adsorbed to the implants surface, and therefore to 2018). Moreover, as this failure is likely to occur at chronic the properties of the material itself (McNally and Anderson, implantation timepoints (months), identification of the problem 2002). While capable of phagocytosing larger particles than their and optimisation of implant design at preclinical testing stages non-fused counterparts, FBGCs release similar degradative and is challenging. Understanding FBR and implementing design chemoattractive factors, and do not appear to play a unique role choices that account for it therefore becomes a crucial step in the in FBR. Their striking morphology, almost exclusive to FBR, is development of medical implants. however a useful tool for the identification of this reaction in While the cellular processes of FBR are well characterized tissue samples in both research and clinic. the properties of an implantable material that initially tag it The combination of macrophages, fibroblasts, and laid down as foreign are not understood. As a result, FBR to implants extracellular matrix build up to create and entirely new tissue— cannot be entirely avoided. Instead, a successful implant capable the FBR capsule. As the capsule develops into a new tissue of carrying out its therapeutic function for its intended lifetime compartment new blood vessels have to extend into it in order needs to accommodate some degree of FBR, and implement to supply it with nutrients. VEGF and PDGF, released by the design choices to ensure that this severity is not exceeded. capsule tissue as it becomes anoxic, act as pro-angiogenic factors drawing in newly-forming blood vessels (Luttikhuizen et al., Implant Design Considerations for FBR 2006). Further cell proliferation and ECM deposition allow the FBR is fundamentally tied to tissue trauma (Wang et al., capsule to progressively thicken over months, until the implant 2015; Klopfleisch and Jung, 2017). Not only is the initial becomes isolated from the surrounding tissue (Figure 2). The implantation of a device the trigger that begins the cellular events capsule eventually reaches a steady-state where macrophage leading to FBR, but subsequent trauma around the implanted activity is no longer intense enough to continue driving fibrosis device leads to further inflammation and worsens ongoing FBR. further away from the implant. The thickness of the capsule and Consequently, one of the most effective strategies to reduce FBR the time needed to reach this point varies greatly, and depends is designing implants that minimize tissue trauma. on multiple factors such as implant materials, size of implant, Implants begin interacting with the body at the moment and location in the body. However, the capsule remains a living they undergo insertion into tissue. While implant design is tissue—since the source of FBR remains present the capsule is often tailored to the location in the body which it will inhabit

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 5 April 2021 | Volume 9 | Article 622524 Carnicer-Lombarte et al. FBR in Nerve Neuroprosthetics for its therapeutic lifetime, it is important to remember that delivering the implant to that location is a key step with consequences for the device’s performance. Tissue damage during implantation is responsible for the initial inflammatory wave attracting cells to the area, and the resulting extravasated blood proteins provide the first interface for the cells to interact with the implant. It is therefore unsurprising that the degree of implantation trauma is linked to the degree of FBR (Wang et al., 2015). While implantation trauma cannot be totally eliminated, implants can be designed accounting for the surgical implantation procedure. For example, some brain probe designs incorporate biodegradable stiff material shanks that enable easy penetration into tissue, degrading thereafter and leaving behind the softer probe (Kozai et al., 2014). Such designs simplify the implantation procedure, eliminating the need for bulkier, more damaging tools which may otherwise be necessary to deliver the probe, and are associated with a lower amount of tissue damage (Kozai et al., 2014). Alternatively, flexible implants can be designed to be combined with purposedly- designed shuttle tools for low trauma implantation into tissue (Figure 3). Implants that take into consideration the mechanics of the implantation procedure and ease of surgical handling can minimize implantation trauma and FBR. Strategies not directly related to the implant itself can also aid in reducing implantation trauma, such as targeting of more easily accessible tissues. Imaging-guided surgical systems can be used to avoid major blood vessels and reduce blood extravasation (Spetzger et al., 1995), optimizing the implantation procedure. Once implanted, devices continue to interact with the surrounding tissue—often in damaging ways. Tissue is subject to continuous motion at various rates and length scales, due to the host’s breathing, blood pumping, movement, and locomotion. While implanted devices will also experience these movements, differences in mechanical properties between implant and surrounding tissue will lead to a mismatch in how the two move and deform in response. As implants fail to move together FIGURE 3 | Implantation of flexible neural probes using stiff shuttles to with tissue, the compression and sliding along the interface minimize trauma and FBR. Reproduced under the terms of the Creative Commons Attribution License (Wei et al., 2018). (A–G) Flexible NET-e devices causes damage to the surrounding tissue (Goldstein and Salcman, containing multiple electrodes. Hole to connect with the shuttle tool seen at 1973; Sharp et al., 2009; Barrese et al., 2016). This not only the bottom of (E). (H–J) Implantation of NET-e devices into the brain using a leads to local inflammation—therefore further exacerbating custom designed and fabricated carbon fiber shuttle device. Entry site FBR—but can also permanently destroy the surrounding target indicated by white arrows. (K–P) Tissue response following long-term (2 month: K–L; 4 month: M–P) implantation of NET-e devices. No significant tissue which may be the therapeutic target of the implant. neuronal death or vasculature disruption seen. Neuronal staining in yellow This mechanical mismatch is particularly relevant for implants (NeuN, K,P). Vascular staining in green (L). Devices seen in red fluorescence containing electronic components, as silicon-based components or pointed by red arrows. Scale bars 50 µm (K–N), 10 µm (O,P). are orders of magnitude stiffer than biological tissues. In order to minimize FBR, implants have begun to experience a shift toward soft or flexible designs, capable of mechanically coupling with Material chemistry is also tightly linked to the body response their local tissue environment. Particularly in neuroprostheses and FBR. Implantable materials need to be carefully chosen to implanted in nervous tissue—some of the softest tissue in the not contain any toxic species which may damage host tissue or body—implants implementing flexible or stretchable materials promote inflammation—a feature generally englobed under the can achieve a much lower degree of FBR and tissue damage term “biocompatible” (Williams, 2008). This not only includes when chronically implanted (Nguyen et al., 2014; Minev et al., material chemistry at the surface of the implant, but also 2015; Lacour et al., 2016; Capogrosso et al., 2018). Smoother components which may leach from within overtime or become implant designs, avoiding sharp corners which can be particularly exposed to the body if surface cracking occurs. Over decades damaging to tissue when motion between the two occurs, can of work, the field of implantable material research has selected also be implemented to decrease tissue damage and chronic materials that exhibit good biocompatibility, including polymers FBR (Veiseh et al., 2015). (e.g., silicones, polyethylene, polyimide), metals (e.g., platinum,

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 6 April 2021 | Volume 9 | Article 622524 Carnicer-Lombarte et al. FBR in Nerve Neuroprosthetics gold) and ceramics. Certain features of material chemistry can Despite its great therapeutic potential, the peripheral nervous also be exploited to beneficially influence FBR, such as the use of system is particularly vulnerable to FBR. Unlike the brain and low-fouling materials which prevent FBR-triggering non-specific spinal cord, which are enclosed in protective bony structure, protein adsorption (Figure 1) (Zhang et al., 2013; Xie et al., nerves run between muscles and organs. Body movements, 2018). A deeper discussion of the interactions between material as caused by normal activities such as locomotion or limb chemistry and FBR, and of the choices of materials in different movements, subject nerves to constant tensile and compressive implants, are beyond the scope of this review and are discussed forces. When an implant is present, these mechanical challenges elsewhere (Williams, 2008; Hassler et al., 2011; Zarrintaj et al., can lead to nerve trauma, exacerbating FBR—particularly when 2018; Mariani et al., 2019). stiff implant materials, contrasting with the very soft nerve In recent decades, more active steps have begun to be tissue, are present (Lacour et al., 2016). Additionally, while the taken to directly tackle FBR. Active strategies meant to directly CNS is considered an immune privileged site (Louveau et al., interfere with the cellular events leading to FBR have been 2015) where inflammation and fibrosis are more subdued than implemented in implantable devices since the incorporation of elsewhere in the body, the peripheral nervous system develops anti-inflammatory compounds in pacemaker leads in the late the typical severity of FBR (Spearman et al., 2018). twentieth century (Mond et al., 2014). Inflammation, however, Nerve neuroprosthetic technologies are very varied, with plays an active role in other biological processes such as repair different designs eliciting different kinds of responses in tissue of tissue damaged around the implantation site, making the (Table 1). The degree of nerve invasiveness is often used as a use of anti-inflammatory compounds not always possible. In metric to group together different classes of neuroprosthetics. recent years, as the finer molecular network underlying FBR are Nerves are complex structures formed by three main types of becoming better understood, new and more specific targets to tissue. The nerve endoneurium houses the axons of neurons, interfere with FBR have become available. This new generation projecting throughout the entire length of the nerve and of active FBR treatments (contrasting with the passive strategies transmitting signals across the body in the form of action relating to implant design discussed above) are discussed later in potentials. The endoneurium is bundled by a tightly packed this review. layer of cells, making the nerve perineurium. While some nerves contain a single of these endoneurial bundles, others contain multiple distinct bundles which are individually referred to as nerve fascicles. Finally, the entire nerve is wrapped in a layer NERVE NEUROPROSTHETICS AND FBR of ECM-rich tissue—the epineurium—which provides structural support and mechanical protection to the nerve (Figure 4). Neuroprosthetic implantable devices deliver a therapy to a Due to this structure, more invasive implant designs with patient by interfacing and interacting with their nervous tissue. electrodes positioned close to axons in the endoneurium This interface is typically electrical in nature—electrodes in can achieve better electrical recording/stimulation, and may the implant record the action potential activity in the nearby selectively target specific subpopulations of axons (e.g., individual neuronal population, and/or activate nearby neurons through fascicles within the same nerve). More invasive implants, electrical stimulation. While potentially able to address a myriad however, can lead to trauma and FBR closer to the fragile nerve of conditions, neuroprosthetics are a class of implantable device axons, potentially leading to the long-term failure of the implant particularly vulnerable to FBR. The fibrotic capsule forming (Figure 5). This invasiveness-selectivity trade-off is a key factor around the implant not only damages and displaces the target influencing the design of nerve neuroprosthetic implants. neuronal tissue, but also forms a high-impedance layer that Based on the degree of invasiveness nerve neuroprosthetic dampens the weak electrical signals produced by neurons designs can be categorized into three different groups and dissipates stimulating electrode currents. FBR is currently (summarized in Figures 4, 5). Epineurial cuff electrodes are the key culprit behind chronically-implanted neuroprosthetic the least invasive, placed around the outside of the nerve without implant failure (Polikov et al., 2005; Salatino et al., 2017), breaching the epineurium. Intraneural penetrating electrodes and poses a major hurdle to the clinical translation of new locally breach the epineurium and perineurium of the nerve, neuroprosthetic technologies. piercing into the endoneurial compartment. Finally, the most Neuroprosthetics have frequently been targeted to the central invasive group are regenerative nerve electrodes, which exploit nervous system, for purposes such as the treatment of Parkinson’s the regenerative properties of the peripheral nervous system in disease and tremor symptoms (Anderson and Lenz, 2006), order to remain embedded in regrowing nerve tissue following restoration of hearing (Colletti et al., 2009), or treatment of an injury. While there is a wide range of nerve implant designs epilepsy (Sprengers et al., 2017). The peripheral nervous system, covering the entire invasiveness-selectivity spectrum, these however, is becoming an increasingly attractive target for the subdivisions provide a useful and commonly used way of delivery of therapies to restore lost or abnormal function. Nerves categorizing implants (Navarro et al., 2005) and discuss their form a fine network of pathways through which the brain implications in the context of FBR. communicates with all structures around the body. By targeting the correct nerve, an implant can activate, modulate, or monitor a Epineurial Cuff Electrodes specific body structure—such as organs (e.g., bladder, pancreas), Cuff electrodes are the simplest type of neuroprosthetic design. muscles (inducing movement), or skin (tactile sensation). Cuff implants typically consist of a hollow cylinder of insulating

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 7 April 2021 | Volume 9 | Article 622524 Carnicer-Lombarte et al. FBR in Nerve Neuroprosthetics scicular Romero et al., 2001 Grill and Mortimer, 2000 Vince et al., 2005 Liu et al., 2019, 2020 Leventhal et al., 2006 Lago et al., 2007 Badia et al., 2011b de la Oliva et al., 2018 Wurth et al., 2017 Christensen et al., 2014 Lago et al., 2005 FitzGerald, 2016 References Cat Rat Rat Cat Animal model 2 2 at 165 at 28 days, 2 2 2 m by 32 weeks Rat µ m Rat µ 0.04 mm 50 0.07 mm 2.13–2.65 mm days ∼ 0.04 mm n.m.∼ ∼ Rat 4.33–4.36 mm n.m. Rat 34 n.m. Rat n.m. Cat area/thickness n.m. Cat n.m. Rat ; PET, Polyethylene terephthalate; LIFE, longitudinally implanted intrafa tic ry st 3 l de of to 12 onic rowth g the y close s eurium. l interface. ocal but chronic ivity. Thin fibrotic capsule ) increased in the nerve ng of the epineurium, without ce) and fibrosis increased in the atch cylindrical cuff. FBR capsule ace between 2 to 6 months by presence of cuff. Abundance of ed to no inflammation or any perceivable n in fascicles penetrated by the array shanks. us classes of nerve neuroprosthetics. developed around inner edge ofbetween cuff. Collagen-rich FBR tissue capsule g and nerve fascicles. inflammation and scar formation around implant. seen surrounding the implant intraneurally. following implantation, reaching a maximumpost-implantation. at A 2 fibrotic weeks capsule developedimplant surroundin and thickening over time. weeks after implantation, but decreasedpost-implantation. by Loss 165 of days axons closetime to points. the implant at chr axonal damage as long as only moderate pressure is applied. post-implantation. Axon numbers declined (axonalmonths death) post-implantation. 6 increased. Reshaping of nerve morphology seen ascapsule soon seen as from 18 7 hours. Fibro daymarkers time at point. 18 Upregulation hours of and inflammato 7 days, which decreased by 1 month. Axon regeneration through microchannels was robustmonths. in This the was fir followed bythickness a of decrease the in fibrotic axon capsule number around as the the inner channel wal abnormally thinly-myelinated axons. Thinning ofExtensive the fibrosis perin in epineurium. FBRcuff. capsule around the insi Nerve tissue and FBR response Fibrotic to the base. Extensive inflammation surrounding the shanks, particularl fibrotic thickening of the nervedeveloped epineurium. extensive Stiff (PET) nerve cuff inflammation andloss fibrosis, of and axons. some have not been included. N.m., FBR capsule thickness not measured in study odes; FINE, flat interface nerve electrode; SELINE, self-opening neura 1 month months period Implant material Implantation Summary and comparison of the nerve response and FBR to vario electrodes; TIME, transverse intrafascicular multichannel electr TABLE 1 | Nerve neuroprosthetic type CuffSpiral cuffSpiral Silicone cuff SiliconeCuff 133–137 days Silicone 28–34 weeksFINE cuff Nerve structure reorganized to m LIFE Nerve morphology deformed Hydrogel 18 hours, or 7 PET days, TIME 6 weeks, Silicone 2 monthsLIFE Soft (hydrogel) cuffs l PolyimideSELINE 3 months Polyimide 3 Parylene months CSlanted Utah array FINE cuffs 2 lead months to a fibrotic thickeni Polyimide UpSieve to Silicon No 32 substantial weeks nerve or axon damage. F No significant loss of Inflammation axons (macrophage orMicrochannel presence Up nerve to act 165 days Up to 350 days Inflammation (macrophage presen Polyimide SiliconeStudies that did Axonal not degeneratio report on the response of tissue following implantation 2 to 12 months 3, 6, and 12 Nerve fiber regeneration took pl

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 8 April 2021 | Volume 9 | Article 622524 Carnicer-Lombarte et al. FBR in Nerve Neuroprosthetics

FIGURE 4 | Overview of different types of nerve neuroprosthetic designs and their implantation locations within the nerve anatomy. (a) Epineurial cuff electrodes, (b) Longitudinally implanted intrafascicular electrodes (LIFE), (c) Transverse intrafascicular multichannel electrodes (TIME), (d) Utah slanted electrode array, (e) Regenerative sieve electrodes, (f) Regenerative microchannel electrodes. material containing one or more pair of electrodes along its The comparatively large size of cuff electrodes and their ease inner surface, which is wrapped around the outermost layer of use have also made cuff electrodes a popular platform for of the nerve (the epineurium) during surgical implantation. the development of new neuroprosthetic-related technologies, Their low invasiveness, simple design and simpler implantation such as wireless ultrasound-driven nerve stimulation (Piech et al., procedure has made them arguably the most successful type 2020), self-wrapping designs (Zhang et al., 2019), and self- of nerve neuroprosthesis in reaching the human clinic. Their healing, stretchable devices that accommodate for tissue growth low invasiveness makes them ineffective at selectively recording as an animal grows (Liu et al., 2020. electrical signals from subsets of axons within a nerve, as Due to the position in which cuff electrodes are implanted, the connective tissue of the epineurium dissipates the low FBR to these cuffs develops predominantly in the epineurium amplitude axon action potentials. Cuff electrodes are, however, of the host nerve. While this is useful to avoid damage to the effective at providing a therapeutic effect through whole nerve fragile neuronal axons in the endoneurium as a consequence of electrical stimulation. inflammation, the epineurium already poses a significant barrier Nerve stimulation carried out through implantable nerve to communication between nerve and electrodes. A thickening cuffs is currently used at the clinical level to treat a variety of of the epineurium as part of FBR can dissipate the already conditions. These include sacral nerve stimulation to restore weak electrical signals transmitted by axons, limiting the use bladder and bowel function (Brindley et al., 1982; Johnston et al., of these devices for electrical recording applications in long- 2005) and peroneal nerve stimulation for the treatment of foot term implantations (Navarro et al., 2005). This effect can be drop (Liberson et al., 1961; Burridge et al., 2007). Electrical compounded when the cuff fits the nerve too loosely. Gaps stimulation of the vagus nerve—a cranial nerve connecting the between the nerve epineurium and implant are prone to filling brain with many of the body’s organs—is also becoming an with fibrotic tissue (Romero et al., 2001), and can increase trauma increasingly popular choice for the treatment of conditions such to the nerve due to mechanical shear damage during movement. as epilepsy (Uthman et al., 2004) and depression (Schachter, While tightly fit cuffs can offer better results, excessive 2002; Nemeroff et al., 2006). Phrenic nerve stimulation, also nerve compression can severely impact long-term nerve known as diaphragm pacing, has also been recently introduced health. Although the nerve connective tissue absorbs tensile into clinical practice as an aid for ventilation in spinal cord injury forces along its axis, compressive forces around the nerve patients (Hirschfeld et al., 2008). are transmitted to the fragile axons in the endoneurium. Outside of the clinic, nerve recordings using cuff electrodes Nerve compression is associated with axonal death and are also being explored as potential therapeutic avenues. inflammation (Krarup et al., 1989; Grill and Mortimer, 2000; Although more challenging to carry out compared to nerve Vince et al., 2005), which can in turn exacerbate FBR to the stimulation due to the distance between electrodes and the implant (Romero et al., 2001). Long-term nerve compression weak electrical signals transmitted by axons, with an ECM-rich can also contribute to the development of neuropathic epineurium separating the two, motor and sensory nerve signals pain (Campbell and Meyer, 2006; Yalcin et al., 2014). gathered using cuff electrodes have been used to guide and Flexible and stretchable materials such as PDMS have been fine-tune prostheses such as artificial limbs in human patients an effective option to build the body of the cuff, able to (Micera et al., 2001; Navarro et al., 2005; Raspopovic et al., 2010). gently squeeze around the nerve epineurium without excessive

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FIGURE 5 | Images of various types of nerve neuroprosthetics. Images included of devices as fabricated (device), implanted into rodent sciatic nerves (implantation), and of the resulting FBR in tissue (FBR). (A-C) Epineurial cuff electrodes. (C) Cross-section of a rat sciatic nerve implanted with a silicone cuff 30 days post-implantation. Nerve (represented by immunoflurescently labeled axon marker NF200, green) take up a small portion of the cuff, with the rest filled with fibroblasts (vimentin, blue) due to FBR to the cuff. Dotted white line indicates approximate position of the cuff. Yellow arrowheads indicate compression of the nerve due to growing fibrotic tissue. Scale bar: 100 µm. (D–F) Intraneural electrodes, either transverse intrafascicular multichannel electrodes (TIME) (D,E) or longitudinally implanted intrafascicular electrodes (LIFE) (F). (F) Toluidine blue-stained images of nerve cross-sections. The two branches of the LIFE parylene C implant (slits in tissue) are found in close proximity to the axons (small circular structures) 1 day (1 d) post-implantation. By 8 weeks post-implantation (8 w) a fibrotic capsule (seen as a smooth ring around the implant slits) develops, displacing the axons. Scale bar: 50 µm. (G–I) Regenerative sieve (G,H) and microchannel (I) electrodes. (I) Cross-section of a rat sciatic nerve regenerated through a PDMS microchannel implant (image of a single microchannel) 12 weeks post-implantation. Nerve tissue (represented by immunoflurescently labeled axon marker NF, green; and Schwann cell S100, red) take up a small portion of the microchannel cross-section. The remainder of the microchannel is filled with other cell types (nuclear stain DAPI, blue), likely macrophages and fibroblasts as a result of FBR to the microchannel walls. The outline of the image represents the approximate position of the microchannel walls. Scale bar: 20 µm. All images reproduced under the terms of the Creative Commons Attribution License: (A) (Christie et al., 2017), (B) (Elyahoodayan et al., 2020), (C) (González-González et al., 2018), (D) (Zelechowski et al., 2020), (E) (Strauss et al., 2019), (F) (de la Oliva et al., 2018), (G,H) (MacEwan et al., 2016), (I) (Musick et al., 2015). compression. From a chemical perspective, PDMS is well (Liu et al., 2019, 2020). However, deformable substrates such as characterized to be inert and biocompatible, not releasing toxic PDMS pose difficulties for the fabrication of electronics (Lacour species of exacerbating the inflammatory response and being et al., 2016). This becomes an increasingly relevant challenge well-suited for long-term implantation (Williams, 2008; Hassler as nerve neuroprosthetics evolve into more elaborate designs, et al., 2011). PDMS is now the material of choice for most nerve aiming to achieve higher resolution communication with the cuffs in clinical practice, including those for vagus nerve (Ben- host nerves. Menachem et al., 2015) and sacral nerve stimulation (Brindley Despite the sensitivity of nerves to poor-fitting nerve cuffs, et al., 1982; Johnston et al., 2005). PDMS cuffs develop lower certain cuff electrode designs purposely apply compressive forces degrees of trauma and FBR than cuffs made from other flexible to the nerve in order to achieve better interfacing. Specifically, materials such as metallic meshes (Christensen and Tresco, the FINE (flat interface nerve electrode) is designed with a 2018), matching the pattern seen in other low-stiffness materials flattened, elliptical bore in order to flatten out large cylindrical

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FIGURE 6 | Bioresorbable electronic patch (BEP) implant for controlled drug release. Reproduced under the terms of the Creative Commons Attribution License (Lee et al., 2019). (A,B) The patches are fabricated from a drug-loaded (doxorubicin) oxidized starch (OST) reservoir, and an electronics-containing compartment made from Mg (conductor), PLGA (dielectric), and PLA (encapsulation) which is bound to it. (C) Wireless control mediates drug delivery into neural tissue. (D-F) Doxorubicin release into tissue occurs over a period of days. (G) The entire implant becomes fully resorbed within 10 weeks of implantation, leaving no adverse reaction in the nearby tissue. nerves. Large sensorimotor nerves often contain axons bundled the axons—often directly inside the endoneurial compartment. into separate fascicles each leading to a different muscle, which The need to penetrate through connective tissue layers in can each be targeted for better therapeutic selectivity. However, order to reach neuronal tissue places similar design needs to fascicles are typically packed tightly together in the nerve cross- those of penetrating electrodes used in the brain, and indeed section, and classical cuff designs struggle to selectively stimulate some intraneural nerve electrodes stem from designs used in individual fascicles. FINE cuffs are designed to flatten the nerve, brain applications. Penetrating electrodes are placed in closer distributing its fascicles over a wider cross-section, making them proximity to axons and without the presence of electrically accessible to sets of electrodes lining the inner surface of the insulating connective tissue layers. Their location in the nerve cuff. This designed has been used to selectively restore sensory anatomy allows them to carry out good quality electrical perception in human arm amputee patients (Tan et al., 2014). recordings, potentially being able to distinguish between signals While excessive compressive forces are associated with axonal from different groups of axons within one same nerve fascicle. death and increased FBR, moderate nerve reshaping with FINE Being embedded within the nerve tissue, penetrating electrodes implants does not seem exert sufficient force to cause axon death are also better anchored to their target tissue which prevents or more connective tissue deposition than seen in wider nerve slippage between the two and ensures that the same groups cuffs (Tyler and Durand, 2003; Leventhal et al., 2006). of axons can be monitored over time. While also useful for Advances in fabrication technologies are also opening new electrically stimulating small groups of axons within the nerve, possibilities for the development of better long-term performing the small size of electrodes and the manufacturing techniques cuff implants. Nerves can be scanned in reconstructed in used in penetrating devices make them less effective at delivering three dimensions in order to later 3D print cuffs perfectly widespread current for whole nerve activation. For nerve adapted to their anatomy (Johnson et al., 2015), potentially stimulation at the clinical level, epineurial cuffs remain the minimizing mechanical trauma and FBR at the nerve-implant preferred choice (Navarro et al., 2005). interface. 3D printed implants can be compatible with electrode Penetrating electrodes, however, face a disadvantage in manufacturing techniques (Haghiashtiani and McAlpine, 2017), the need to rupture through nerve compartments during opening the door for future perfect-fit epineurial cuff electrodes implantation. This process not only generates more trauma than suitable for clinical use. an epineurial device, but also localizes the trauma closer to axons. This can not only lead to the development of a fibrotic capsule Intraneural Penetrating Electrodes deep within the nerve tissue, but can also lead to nearby axon Intraneural electrodes are designed to penetrate the outer layers death due to the inflammation associated with FBR. Moreover, of the nerve and position the electrodes in closer proximity to by remaining implanted within the nerve tissue, shear damage

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 11 April 2021 | Volume 9 | Article 622524 Carnicer-Lombarte et al. FBR in Nerve Neuroprosthetics due to mechanical mismatch between the two becomes a major 2020). Self-deploying anchors can also be incorporated into source of further trauma that can potentially worsen axon death intraneural designs, minimizing movement and the associated and fibrosis. trauma following implantation, as seen with self-opening While intraneural penetrating electrode designs have not neural interface (SELINE) devices (Cutrone et al., 2015), and yet reached widespread use in the human clinic, a wide generating minimal FBR (Wurth et al., 2017). Combined with range of designs have been developed over the years in the potential improvements in the implantation procedure, flexible research environment. Longitudinal-interfascicular electrodes penetrating nerve implants such as these hold great potential for (LIFE), consisting of a long flexible body with a single electrode long-term therapeutic applications and translation into clinical implanted into the endoneurium of a nerve fascicle with the aid use, as highlighted by the several recent uses of TIMEs in human of a rigid needle, were one of the earliest penetrating designs patients (D’Anna et al., 2019; Petrini et al., 2019). developed (Malmstrom et al., 1998). More recently, updated Most of the materials used in these flexible devices LIFE designs manufactured using microfabrication technology are chosen to have well-established biocompatibility—being and containing multiple electrodes along its length have also chemically inert to the body and remaining so for long been developed (thin-film LIFE) (Navarro et al., 2007). LIFE periods of implantation—while also being compatible with thin- implants are effective for long-term recording and stimulation film fabrication techniques. Polyimide is particularly popular applications, having for example been used in humans for for nervous system implant applications—including the PNS. the recording of volitional limb movement and to elicit skin Polyimide is biocompatible and exhibits low cytotoxicity in vitro, sensation of touch/pressure (Dhillon et al., 2004, 2005; Micera and remains stable for long periods of implantation (Richardson et al., 2008). et al., 1993; Rubehn and Stieglitz, 2010; Hassler et al., 2011). While LIFE implants are limited to interfacing with a single Parylene C has recently arisen as a good material option for nerve fascicle, transverse intrafascicular multichannel electrodes peripheral nerve devices (de la Oliva et al., 2018), and has a well- (TIMEs) are designed to penetrate through the entire nerve established history of biocompatibility and biostability (Chang cross-section and establish connection with multiple nerve et al., 2007; Winslow et al., 2010; Hassler et al., 2011). fascicles. By stimulating one or multiple fascicles simultaneously, Stiff material penetrating arrays have also seen widespread use TIMEs are able to produce a wider range of distinct nerve as nerve neuroprosthetics. Utah arrays were developed in the activation patterns. This can, for example, be used to reproducea early 1990s as a means to deploy large numbers of electrodes wider range of distinct sensory perceptions in amputee patients to deep layers of the brain (Normann and Fernandez, 2016), (Strauss et al., 2019). TIME implants are also fabricated from and have since been adapted for use in nerves (Branner and flexible materials such as polyimide, and are also inserted with the Normann, 2000; Branner et al., 2001). These implants consist of aid of a microneedle which is removed after surgical implantation arrays of stiff silicon shanks with individual electrodes exposed (Boretius et al., 2010). TIMEs are able to both stimulate (Badia at their tips. During implantation the shank array is pierced et al., 2011b) and record from Badia et al. (2016) separate nerve directly into the neural tissue. The variety fabricated for use in fascicles within nerves independently, and coupled with artificial nerves has shanks of variable depth, allowing electrodes to be hand prostheses have been used to restore sensory perception in deployed into different fascicles and giving them the name of human hand amputee patients (Raspopovic et al., 2014; D’Anna slanted Utah arrays. In contrast to other penetrating probes, Utah et al., 2019). Stimulating TIMEs have also been used to restore arrays incorporate up to 100 electrodes which can be deployed sensory perception in leg amputees, combined with pressure across the entire cross-section of the nerve—ensuring that the sensor-equipped leg prosthetics (Petrini et al., 2019). entire nerve axon population can be interfaced with. Slanted LIFE and TIME nerve neuroprosthetics share a key similarity Utah arrays have been used for both stimulation (restoration of in their design: the use of flexible materials. Flexible implants sensory perception) and recording (monitoring of motor activity) are able to better deform with the nerve tissue, as the two move applications in human hand amputee patients (Clark et al., 2014; around together during normal body activity. Flexible implants Davis et al., 2016; Wendelken et al., 2017). Nerve recordings with in general show improved tissue compatibility compared to these implants were able to guide the movement of a virtual stiffer equivalents, minimizing trauma and FBR arising from hand prosthesis over a period of 30 days, indicating that robust shear damage (Lacour et al., 2016; Salatino et al., 2017). This recordings can still be achieved during the onset of the chronic is particularly relevant for soft and fragile tissues, or tissues stages of FBR (Davis et al., 2016). which are constantly exposed to movement—two characteristics Despite the success of Utah arrays in producing high quality which apply to the peripheral nervous system. Though LIFE multi-site recordings in both CNS and PNS, from a long- and TIME implants still induce a substantial amount of trauma term stability and FBR perspective these implants are more during the implantation procedure, they elicit a limited degree problematic than other penetrating designs. While as a material of FBR. While a thin fibrotic capsule forms around these flexible silicon is chemically inert and biocompatible, permitting cell devices over chronic implantation time points, the degree of growth and not causing particular adverse reactions in tissue over fibrosis is mild enough to permit stable long-term recordings, long implantation periods (Bayliss et al., 1999; Shin et al., 2019), with no widespread inflammation or axon damage seen (Lago its high stiffness leads to poor tissue compatibility when applied et al., 2007; Badia et al., 2011a). The mechanical matching with to such an invasive format. Utah arrays rupture nerve tissues tissue renders these implants very robust, exhibiting very little when implanted for each of the many electrodes it contains, damage or delamination after implantation (Cvancaraˇ et al., greatly increasing insertion trauma. Moreover, their stiff material

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 12 April 2021 | Volume 9 | Article 622524 Carnicer-Lombarte et al. FBR in Nerve Neuroprosthetics body contributes to greater mechanical shear damage around of axons over a 3 month period, likely a consequence of the each of the shanks, worsening long-term trauma and FBR. implantation trauma combined with subsequent damage caused This mechanical damage is severe enough to damage not only by the proximity of the implant walls to the fragile axons. Their the fragile nervous tissue, but also the probes themselves. In good recording properties and ease of use have, however, found the CNS, a tissue less exposed to movement than peripheral use for them in ex vivo applications such as nerve-on-a-chip nerves, a study determined that 90% of Utah arrays implanted platforms (Gribi et al., 2018). in non-human primates failed over a 6 year chronic implantation period, with most of failures seen within a year of implantation Regenerative Nerve Electrodes (Barrese et al., 2016). Slanted Utah arrays implanted into nerves The last class of implants are regenerative nerve electrodes, cause large amounts of tissue damage, which over long-term which exploit the regenerative capacity of peripheral nerves in implantation periods lead to larger FBR responses and chronic order to embed electrodes deeply into nerve tissue. In contrast inflammation (Christensen et al., 2014, 2016). to the central nervous system, peripheral nerves are capable of Slanted Utah arrays continue to be of great value as research undergoing some degree of regeneration when injured. When tools, and show promise for the translation of new therapies a nerve is damaged axons past the lesion site degenerate, while to the clinic. Recently, slanted Utah arrays implanted into the axons proximal to the lesion begin regrowing across it in an median and ulnar nerves of a hand amputee patient. Through attempt to reconnect with their distal target. If an implant stimulation of the nerves, and in combination with muscle containing electrodes is introduced into a nerve lesion, these recordings, the patient was able to drive and receive sensory axons will often grow through the implant as part of their feedback from a bionic hand (George et al., 2019). Despite regenerative response. This strategy can be used to position large this and other prior translational successes (Clark et al., 2014; numbers of electrodes throughout the entire cross-section of the Davis et al., 2016; Wendelken et al., 2017), the large degree of nerve and, once the regenerating axons reconnect to their targets trauma and FBR associated with current Utah array designs poses at the distal end of the nerve, the array of electrodes can be used difficult challenges for their widespread clinical use. stimulate or record the electrical activity of small axon subsets. While most penetrating electrodes are designed to While regenerative implants are effective at tightly interfacing pierce directly into the endoneurial compartment, certain large numbers of electrodes with a nerve, they are also designs employ a less invasive approach. Slowly penetrating associated with the largest amount of implantation trauma. interfascicular nerve electrodes (SPINEs), for example, consist Whether occurring accidentally or purposely induced (in of a nerve cuff incorporating penetrating electrode-equipped research models), regenerative implants require a nerve lesion flaps over its inner surface, which slowly penetrate into the to be present. Most commonly this is in the form of a full nerve cross-section after implantation. While piercing into the nerve transection, where the two ends of the nerve are cleanly ECM-rich epineurium, these flaps settle between fascicles and do separated and a gap forms between the two (Navarro et al., 2005; not pierce the perineurium. By bypassing part of the epineurium, Thompson et al., 2015). Lesioning brings with it large amounts these electrodes achieve a decrease in stimulation threshold of inflammation and fibrosis which, although initially directed and can achieve better selectivity than traditional epineurial toward guiding axon regeneration and repairing the nerve tissue, cuff designs (Tyler and Durand, 1994, 1997). Although SPINE can later worsen FBR around the implant. designs achieve some of the advantages of intrafascicular designs, The first widely used type of regenerative nerve they can exhibit better long-term stability by localizing fibrosis neuroprosthetics to be developed were sieve electrodes. and inflammation due to FBR to the outer, more robust, layers of Early sieve electrode designs were fabricated from rigid silicon the nerve architecture. wafers perforated with an array of holes, each of which contained On the other end of the spectrum, certain types of nerve an electrode (Edell, 1986; Akin et al., 1994; Navarro et al., neuroprosthetics achieve fascicle recordings by more severely 1996). Silicon is bioinert as a material, permitting cell growth disrupting the tissues of the nerve. Microchannel implants— (Bayliss et al., 1999) and remaining stable for long periods of devices made up of multiple electrode-containing channels implantation without causing adverse tissue reactions (Shin designed to each host a subset of the nerve’s axons, and et al., 2019). However, the high stiffness of silicon can lead to more commonly used as regenerative nerve electrodes—have mechanical damage surrounding silicon implants. Silicon sieves also been used to interface with healthy nerves. Implantation were observed to lead to extensive axon death after implantation of these devices involves the surgical disruption of the nerve and were replaced by more flexible materials such as polyimide epineurium, followed by the gentle teasing of the axons within in later designs (Shimatani et al., 2003; Ramachandran et al., into small bundles which are then enclosed within the implant’s 2006). Upon implantation into a transected nerve axons would channels. While the severe implantation trauma caused by the regenerate through the sieve’s holes, allowing stimulation or full dissection of the epineurium can lead to axon damage, monitoring of their electrical activity. While holes with diameters certain nerves are able to support this implantation procedure as small as individual axons (2–10 µm) could theoretically have (Chew et al., 2013). Such microchannel electrodes have, for allowed individual recordings of single regenerated axons, axon example, been implanted in nerves at locations very proximal regeneration fails through such small holes (Navarro et al., 1998). to the dorsal root ganglia to monitor bladder function in Instead, holes of 40–65 µm were typically preferred (Navarro rodents at long-term implantation time points (Chew et al., et al., 1998). Sieve electrodes implanted into regenerating nerves 2013). However, the implanted nerves experienced a steady loss in rodent models have been successfully used to record gustatory

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(Shimatani et al., 2003) as well as sensorimotor (Ramachandran 2006). The channels in microchannel devices are, however, orders et al., 2006) signals weeks after implantation. More recently, of magnitude longer than the holes of a sieve electrode. This additional features such as the incorporation of porous “transit” creates a need to fully vascularise the tissue within the channels zones in the center of the sieve and the use of glial derived to supply the axons with nutrients and prevent their death, neurotrophic factor (GDNF) to improve nerve regeneration and which makes them more vulnerable to the slow build-up of survival have been implemented (MacEwan et al., 2016). fibrotic tissue during chronic FBR. While axon regeneration can Aside from the injury-associated trauma related to the occur in microchannel devices with channels as small as small implantation of a regenerative implant, sieve electrodes are also as 55 µm in diameter (similar to sieve electrode dimensions), vulnerable to further long-term tissue trauma and FBR. The optimal axon regeneration is seen when coupled with growth narrow holes through which axons regenerate become a choke of one or more blood vessel, requiring larger microchannels of point where trauma and fibrosis can easily develop. Implanted 100 µm diameter (Lacour et al., 2009). Once regeneration has sieve electrodes made of polyimide experience a steady increase occurred, however, the progressive narrowing of the channel in axonal numbers over the first 6 months post-implantation, diameter as FBR develops over its inner wall leads to the eventual as all axons slowly regenerate through the implant and into occlusion of these blood vessels, depriving axons of their nutrient the distal nerve stump. This is, however, followed by a sharp supply. By 6 months post-implantation PDMS microchannel decline in axon numbers at the 12 month time point (Lago devices can develop capsules within the channels 28 µm in et al., 2005). Inflammation, characterized by an accumulation thickness which, while still leaving a large portion of the channel of macrophages around the edges of the sieve holes, appears diameter unoccluded, lead to the death of the majority of the to peak 6 months into implantation followed by a moderate axons (FitzGerald, 2016). decline—possibly as the holes become filled with fibroblasts and While sieve and microchannel electrodes are the most distinct ECM (Klinge et al., 2001). regenerative nerve designs, specifically developed to be used in A more recent evolution of the sieve electrode design are regenerating nerves, many other classes of implantable nerve microchannel electrodes, composed of a solid cylinder of material neuroprosthetics can be used as regenerative devices. Cuff perforated by an array of long channels, each housing within electrodes can be inserted into the gap of a nerve lesion and them one or more electrodes. Microchannel devices exploit the the nerve allowed to grow through it, rather than wrapping the fact that fully transected nerves are capable of regenerating cuff around a healthy nerve (Lotfi et al., 2011; Stoyanova et al., across up to centimeter-long gaps left between the nerve 2013; Thompson et al., 2015). Regenerative designs ensure that stumps (Deal et al., 2012). Microchannel devices are typically tissue always perfectly surrounds the implant, providing tighter several millimeters in length, and have channels of ∼100 µm interfacing and higher quality electrical stimulation/recording diameter. While microchannel electrodes might at first look properties without accidentally excessively compressing the like a longitudinally-extended version of the sieve electrode nerve. Similarly, cylindrical conduits implanted into a gap lesion design, microchannels offer a number of advantages. The walls can also be equipped with penetrating implants facing its bore, of the microchannels—made from insulating materials—serve allowing axons to regrow around them for better interfacing to amplify the weak axon electrical signals, and shield the (Musallam et al., 2007; Clements et al., 2013). Thin-film devices electrodes from external noise (Fitzgerald et al., 2008). While can also be incorporated into bridging conduits to split the originally made from polyimide (FitzGerald et al., 2012), more bore into two or more channels, achieving some of the higher- recent microchannel designs are built from stretchable and selectivity benefits provided by microchannel designs (Delgado- softer materials such as PDMS (Musick et al., 2015; Lancashire Martínez et al., 2017). Although these devices may perform better et al., 2016). Microchannel devices have been used to record than their non-regenerative counterparts, the large amount of sensorimotor nerve signals in rodent models (Gore et al., trauma and subsequent inflammation and FBR are unlikely to be 2015; Srinivasan et al., 2015b), and have been shown to work worthwhile trade-off unless a nerve lesion has already occurred. even in absence of a regeneration target in full amputation models (Srinivasan et al., 2015a). While many of the same implications for FBR that apply sieve electrodes also apply to microchannel electrodes, microchannels LOOKING AHEAD—NEW MATERIALS AND both offer some advantages and face additional challenges. On STRATEGIES TO MINIMIZE FBR one hand, microchannel devices are often made from stretchable materials, such as PDMS. These materials allow the channel The development of a foreign body reaction in response to walls to deform, in contrast to sieve polyimide sieve electrodes the implantation of materials into nerves has been reported in which can only bend within the nerve cross-sectional plane. This literature for decades (Kim et al., 1976). Over the years, a wide can minimize the degree of long-term damage caused to the range of nerve neuroprosthetic designs have been developed and nerve tissue within the channels, decreasing FBR and axon death. tested. This has provided a wealth of data on the advantages Studies have achieved long-term recordings in freely moving and disadvantages of different designs, including their effect animals using microchannel devices for periods 4 months post- on FBR and tissue trauma. While a lot can be learned from implantation (Gore et al., 2015), while only small signals in past designs for the development of implants with better long- response to electrical stimulation have been recorded at such term stability and tissue compatibility, there has also been an chronic timepoint with sieve electrodes (Ramachandran et al., increasing interest in gaining a deeper understanding of the

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 14 April 2021 | Volume 9 | Article 622524 Carnicer-Lombarte et al. FBR in Nerve Neuroprosthetics

TABLE 2 | Summary of factors influencing FBR to implants and strategies et al., 2017). Interference with cell adhesion can also be achieved employed to minimize it. through chemical means. Low fouling materials minimize the

Implant properties and Strategies to minimize FBR adsorption of proteins to their surface—the first step leading downstream effects to FBR—and coating implants with these can greatly diminish influencing FBR FBR (Xie et al., 2018). Material stiffness also greatly contributes to FBR, with Chemical biocompatibility • Bioinert and long-term stable materials. softer materials (closer in stiffness to that of biological and material degradation • Degradable materials that are removed from the body before FBR progresses into its tissues) generating less mechanical trauma and therefore less chronic stage. FBR (Lacour et al., 2016; Salatino et al., 2017). However, Biofouling of the implant • Low fouling materials or coatings at the similarly to implant size, stiffness also appears to play a implant surface. direct role in guiding FBR independently of the mechanical Implant size and shape • Microfabrication of smaller implants and use damaged caused during movement. Nerve implants coated of designs better suited for their with a range of very low stiffness materials develop less nerve environment. fibrosis and inflammation than those coated with less soft Implant stiffness • Soft and flexible materials or materials (Carnicer-Lombarte et al., 2019). How properties implant coatings. such as size, geometry and stiffness directly guide FBR is not Tissue inflammation • Anti-inflammatory compounds, particularly understood. While no specific mechanism has been identified, locally delivering through the implant. • Compounds targeting mechanotransduction (the conversion of mechanical cues into FBR-specific inflammation. biochemical signals) is thought to play a role. Cells such as Fibrosis and capsule • Anti-fibrotic compounds. macrophages are known to actively interact with their mechanical formation • Suppressants of vascular growth. environment and modulate their inflammatory activity in response (Iskratsch et al., 2014). Note certain factors contributing to FBR have more than one strategy FBR-reduction strategy associated. Pharmacological therapies also have great potential to target the downstream consequences of FBR. FBR is primarily mediated by inflammation—macrophages respond to the causes and pathways underlying FBR—and ways of directly presence of the implant and orchestrate the fibrotic response tackling this reaction (Table 2). that leads to its encapsulation. Anti-inflammatory compounds FBR occurs in response to any material that is implanted can directly inhibit FBR by dampening this inflammatory into the body (Anderson et al., 2008; Salatino et al., 2017). response. A decrease in macrophage activity around the However, certain material properties can exacerbate or decrease implant decreases both their adherence and proliferation FBR. Implantable materials must exhibit good biocompatibility, around the implant and the recruitment of capsule-forming being chemically inert to tissue to minimize inflammation and fibroblasts (Figure 1). Dexamethasone—a commonly used cell death (Williams, 2008; Hassler et al., 2011; Mariani et al., anti-inflammatory corticosteroid drug—has found great success 2019), and remain so for the necessary long implantation in pacemaker stimulator implants used in the clinic, where it periods. Even if used materials are chosen to be biocompatible, is incorporated into the material surrounding the electrodes mechanical factors such as the size and geometry of implanted and elutes over time into the surrounding tissue to hamper bodies fabricated from these also has a direct impact on the FBR and improve implant life-time and stability (Mond et al., severity of the FBR. Trauma is intimately tied to FBR, as the 2014). Dexamethasone has also been tested for control of inflammation resulting from tissue damage will attract immune FBR in nerve neuroprosthetics. Regenerative microchannel cells to the area which can join the reaction against the implant implants incorporating dexamethasone show a decrease in (monocyte/macrophage recruitment step, Figure 1). Different the thickness of the fibrotic capsule and avoid the death of size and shape implants will trigger different amounts of FBR axons over time seen in un-treated implants (FitzGerald, based on the degree of mechanical trauma they cause in when 2016). Dexamethasone administered systemically has also been moving in their implanted location. However, even when these shown to improve the electrical stimulation properties using variables are eliminated—for example by implanting a mass of TIME implants over a 3-month implantation period (Oliva spherical implants of different sizes into the peritoneal cavity of et al., 2019). Despite its effectiveness, dexamethasone hampers rodent models—size appears to directly impact on FBR. Spherical all processes mediated by inflammation—including tissue implants of 0.5 mm diameter generate higher degrees of FBR healing. Dexamethasone use can limit tissue healing following than larger 1.5 mm ones, an effect which is consistent for a wide implantation trauma and the chronic trauma associated with range of materials (Veiseh et al., 2015). Similarly, shape appears the indwelling implant, which can be particularly detrimental to directly influence FBR, with sharper edges generating more in regenerative implants relying on the regrowth of nerve tissue fibrosis than spherical implants (Veiseh et al., 2015). Very small into the implant (FitzGerald, 2016). Similar to dexamethasone, implants—with dimensions smaller than individual cells—such other general anti-inflammatory drugs such as aspirin (Malik as injectable mesh electrodes seem to generate minimal amounts et al., 2011) and rapamycin (Takahashi et al., 2010) have also been of FBR, potentially largely bypassing this response by preventing shown effective for the reduction of FBR. However, although cells (neutrophils, macrophages and fibroblasts in Figure 1) from the pathway underlying FBR in inflammation has not been fully spreading on their surface and becoming activated (Zhou characterized, certain mediators are beginning to be identified

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 15 April 2021 | Volume 9 | Article 622524 Carnicer-Lombarte et al. FBR in Nerve Neuroprosthetics and provide new pharmacological targets against FBR. CSF1 Rogers and colleagues have designed and fabricated a class receptor has been recently identified as playing a unique role of biodegradable sensors to monitor relevant physiological in FBR-associated inflammation, with its inhibition using both parameters such as cerebral temperature and oxygenation (Bai implant-encapsulated and systemically-delivered small molecule et al., 2019), spatiotemporal neural activities in the cerebral cortex compounds leading to a decrease in macrophage activity and (Yu et al., 2016), and intracranial pressure (Kang et al., 2016). fibrosis around implanted materials without impacting wound Biodegradable therapeutic devices such as electronic stents for healing (Doloff et al., 2017; Farah et al., 2019). controlled drug releases for endovascular diseases (Son et al., Further downstream in the FBR process, fibrosis can also 2015), or wireless tissue heating for infection treatment (Tao be targeted as a means to eliminate some of the more et al., 2014) both demonstrated the high specificity and efficacy severe consequences of FBR (fibrotic encapsulation, the last of targeted implantable devices while avoiding the negative tissue step of FBR—Figure 1). TGF-β lies at the heart of the encapsulation due to FBR. fibrotic response in FBR, driving the activation of fibroblasts However, some studies still reported moderate degrees of surrounding the implant into myofibroblasts (Ignotz and FBR at the implantation sites of biodegradable bioelectronics Massagué, 1986). Local inhibition of TGF-β through injection (Xue et al., 2014). These studies postulated that the FBR might of anti-sense oligonucleotides has been shown to decrease be due to incomplete degradation of the polymeric materials, capsule formation in subcutaneous implants (Mazaheri et al., and/or the accumulation of debris after the materials are 2003). Suppressors of TGF-β are entering the clinic for the degraded. As a result, careful design and modulating between management of fibrotic diseases, offering an opportunity for the active/encapsulating layers of the biodegradable electronics their use in FBR (King et al., 2014). Growth of the fibrotic is essential to the success of mitigating FBR in this class of capsule also requires the extension of new blood vessels implantable electronics. into the newly forming tissue. Local scavenging of VEGF around implants, a stimulator of endothelial growth released by anoxic tissue, can inhibit the extension of new blood vessels THE FUTURE OF NERVE necessary to support the fibrotic capsule and decrease fibrotic NEUROPROSTHETICS encapsulation (Dondossola et al., 2017). Apart from surface modification or adjusting the invasiveness Despite its unavoidable presence, FBR has until recently of the implants, devices made of biodegradable and bioresorbable been an acceptable side effect of the implantation of nerve materials have been increasingly gathering momentum as a neuroprosthetics. In the clinic, nerve cuffs have bypassed its effect viable class of bioelectronics to minimize the effect of FBR by delivering larger stimulating currents to entire nerves, while in when implanted (Veiseh and Vegas, 2019) (Figure 6). The the research environment shorter implantation time points have fibrotic components of FBR develop to chronically-indwelling allowed the development of implantable technologies without implants. Biodegradable implants prevent the development of allowing fibrosis to develop. However, nerve neuroprosthetics fibrosis by allowing the acute inflammatory phases of FBR to continue to evolve with advances in knowledge of materials degrade part or the entirety of the implant once its therapeutic and manufacturing techniques, leading to higher resolution role is complete. Biodegradable and bioresorbable devices designs requiring more intimate interfacing with neuronal offer a unique opportunity to simultaneously perform targeted tissues. Together with a growing interest in the translation of diagnosis and therapy in vivo with minimal long-term FBR these technologies into human clinical use (Wendelken et al., consequences (Grossen et al., 2017). 2017; D’Anna et al., 2019; Petrini et al., 2019), FBR has become For applications where the functions of the implants are a dominant problem that needs to be actively addressed at the only necessary for a predefined period of time, this class of implant design stage. so-called transient electronics employ a layered design (Hwang Since its infancy, the field of nerve neuroprosthetics has et al., 2012) where degradable polymeric materials act as overall transitioned from stiff materials, well suited for traditional an encapsulation layer, with functional inorganic materials microfabrication techniques, to flexible devices. While cuffs have such as zinc, magnesium, or silicon enclosed inside. While been the predominant choice for widespread clinical stimulation the active layers can remain long-term in vivo (producing a applications, newer ultraflexible devices such as the TIME are much smaller, lower trauma-inducing device than the original seeing great success in humans (D’Anna et al., 2019; Petrini implanted one), they can also themselves be degradable. et al., 2019). The success of these devices can serve to inspire Not only does the degradable device prevent any need the design of new flexible nerve implant designs and drive a for later surgical extraction once the device lifetime has push for the translation of existing ones toward human use. expired (Mattina et al., 2020), biodegradable devices also CNS neural interfaces are experiencing a similar shift toward provide an alternative route for the immune system to soft/flexible designs (Salatino et al., 2017). Low FBR designs process the device and lead to a much lower degree of developed for brain applications may be adapted for use in FBR. Both the degradation time and rate of degradation peripheral nerves, such as injectable mesh electrodes (Zhou can be adjusted by modulating the thickness between the et al., 2017) or ultraflexible non-penetrating transistor arrays active and encapsulation layers, allowing the devices to remain (Khodagholy et al., 2013). Neural tissue is some of the softest in in vivo from days, weeks, to months depending on the the body (Cox and Erler, 2011), and remains orders of magnitude applications (Choi et al., 2016). softer than many materials commonly regarded as soft (Lacour

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 16 April 2021 | Volume 9 | Article 622524 Carnicer-Lombarte et al. FBR in Nerve Neuroprosthetics et al., 2016), leaving ample room for the improvement development of new-generation nerve neuroprosthetics. Other of current soft designs into even more biocompatible and novel strategies, such as the incorporation of cells or tissue within FBR-minimizing ones. devices to transform them into living implants, interacting with A transition to soft nerve neuroprosthetics will bring with and integrating into the host (Rochford et al., 2020), may also it fabrication challenges. The field of neuroprosthetics has provide unique avenues to address FBR. While the inclusion of greatly benefitted from microfabrication technology used in these additional modalities introduces further challenges for the the semiconductor industry, which does not easily translate to manufacturing of neuroprosthetics, the therapeutic potential of soft or flexible substrates. This becomes particularly relevant implantable devices capable of stable, fine resolution recording as implant designs become smaller in scale and increasingly and stimulation over decades far outweigh the costs. elaborate in order to record signals from small subsets of axons within a nerve. A range of strategies are being developed DATA AVAILABILITY STATEMENT with this objective, such as the use of stiff but also thin and therefore flexible materials (Khodagholy et al., 2015), The original contributions generated for the study are included the engineering of defects in stiff materials to render them in the article/supplementary material, further inquiries can be deformable (Vachicouras et al., 2017), or the encapsulation of directed to the corresponding author/s. electronics in soft materials (Liu et al., 2019). An alternative—or more likely a complementary—strategy for AUTHOR CONTRIBUTIONS future nerve neuroprosthetic designs will be the incorporation of compounds or materials that actively target FBR. Several AC-L and DB designed the paper and analyzed the FBR-targeting compounds are already progressing toward use literature. AC-L and S-TC wrote the paper. GM and in humans (King et al., 2014; Farah et al., 2019), and as our DB revised the paper. All the authors read and approved understanding of FBR continues to evolve this repertoire will the manuscript. likely continue to expand. Future nerve neuroprosthetics will likely be multimodal, containing not only electrode arrays to FUNDING electrically communicate with tissue, but also mechanisms to chemically interact with it in the form of FBR-modulating This work was supported by the Engineering and Physical compounds. The delivery of these compounds may occur Sciences Research Council (EPSRC; 27 EP/S009000/1). through engineered structures such as microfluidic channels, AC-L acknowledges support from the Wellcome Trust or provided through material-oriented solutions such as (Wellcome Trust Junior 28 Interdisciplinary Fellowship). immobilization of compounds on their surface or encapsulation S-TC acknowledges support from the Cambridge Trust. DB within materials. The field of surgical nerve repair already is supported by Health Education England and the National employs implants actively modulating biological processes Institute for Health Research HEE/ NIHR ICA Program Clinical (Carvalho et al., 2019), and may be a useful source for the Lectureship (CL-2019-14-004).

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Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 21 April 2021 | Volume 9 | Article 622524 Carnicer-Lombarte et al. FBR in Nerve Neuroprosthetics

Zarrintaj, P., Saeb, M. R., Ramakrishna, S., and Mozafari, M. (2018). chronic immune response in the brain. Proc. Natl. Acad. Sci. 114, 5894–5899. Biomaterials selection for neuroprosthetics. Curr. Opin. Biomed. Eng. 6, doi: 10.1073/pnas.1705509114 99–109. doi: 10.1016/j.cobme.2018.05.003 Zelechowski, M., Valle, G., and Raspopovic, S. (2020). A computational model Conflict of Interest: The authors declare that the research was conducted in the to design neural interfaces for lower-limb sensory neuroprostheses. J. absence of any commercial or financial relationships that could be construed as a NeuroEngineering Rehabil. 17:24. doi: 10.1186/s12984-020-00657-7 potential conflict of interest. Zhang, L., Cao, Z., Bai, T., Carr, L., Ella-Menye, J.-R., Irvin, C., et al. (2013). Zwitterionic hydrogels implanted in mice resist the foreign-body reaction. Nat. Copyright © 2021 Carnicer-Lombarte, Chen, Malliaras and Barone. This Biotechnol. 31, 553–556. doi: 10.1038/nbt.2580 is an open-access article distributed under the terms of the Creative Zhang, Y., Zheng, N., Cao, Y., Wang, F., Wang, P., Ma, Y., et al. (2019). Commons Attribution License (CC BY). The use, distribution or reproduction Climbing-inspired twining electrodes using shape memory for peripheral in other forums is permitted, provided the original author(s) and the nerve stimulation and recording. Sci. Adv. 5:eaaw1066. doi: 10.1126/ copyright owner(s) are credited and that the original publication in this sciadv.aaw1066 journal is cited, in accordance with accepted academic practice. No use, Zhou, T., Hong, G., Fu, T.-M., Yang, X., Schuhmann, T. G., Viveros, R. D., et al. distribution or reproduction is permitted which does not comply with these (2017). Syringe-injectable mesh electronics integrate seamlessly with minimal terms.

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